WO2001011051A2 - Chimäre proteine - Google Patents
Chimäre proteine Download PDFInfo
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- WO2001011051A2 WO2001011051A2 PCT/DE2000/002657 DE0002657W WO0111051A2 WO 2001011051 A2 WO2001011051 A2 WO 2001011051A2 DE 0002657 W DE0002657 W DE 0002657W WO 0111051 A2 WO0111051 A2 WO 0111051A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to recombinant chimeric proteins which have a nucleic acid synthesis activity, complexes containing them, nucleic acids coding for them, vectors and cells containing them, an antibody directed against them, uses of these proteins, kits containing them and methods for elongation, amplification, reverse transcription, Sequencing and labeling of nucleic acids.
- proteins are not present in the cell as monomers, but are part of a functional multimeric complex. Examples of such complexes are described in almost all areas of cell biology (e.g. transcription, translation, replication, cytoskeleton, signal transduction, mRNA processing).
- interaction or binding of a protein with or to another protein takes place via certain amino acids or amino acid sequences, which are mostly on the surface of the protein in the folded protein and for specific binding or interaction with or with are responsible to the partner.
- This amino acid or such an amino acid sequence is referred to hereinafter as the “interaction-dependent sequence” or interaction-mediating sequence.
- the amino acids which are involved in the formation of an interaction do not necessarily follow one another directly in the primary amino acid sequence, but instead may be responsible for more or less conserved positions within the amino acid sequence of the protein or peptide.
- donor proteins are to be understood as those proteins which interact with another protein and optionally bind another protein.
- acceptor proteins are understood to be those proteins which interact with another protein and optionally bind another protein
- the interaction-related sequence of a protein-protein interaction site includes (i) the determination of the three-dimensional structure of a complex of donor and / or acceptor protein by X-ray structure analysis or (ii) NMR methods.
- a further method is (iii) to reconstitute a complex binding in vitro with recombinant proteins and to determine the interaction-dependent sequence (s) by targeted modification of the donor or acceptor protein.
- targeted changes include the mutation of individual amino acids, for example to alanine (alanine scanning) or the deletion of sequences in the protein.
- the interaction-dependent sequence can be defined in this way.
- Y2H Another method for determining the interaction-dependent sequence of a protein is based on the use of a two-hybrid system, hereinafter also abbreviated as "Y2H".
- Y2H systems are based on the fact that you tein with a detectable activity (such as the enzyme "dihydrofolate reductase") expressed as two non-covalently linked parts. This protein is inactive if the two parts are not in close proximity to each other .
- the two proteins to be examined are each formed with a fusion protein with one of the two parts of the protein with the detectable activity (such as dihydrofolate reductase) fused and expressed so that two fusion proteins are formed.
- the detectable activity such as dihydrofolate reductase
- Enzymes dihydrofolate reductase, beta-galactosidase
- signal transduction proteins Cdc25 from Saccharomyces cerevisiae
- transcription activators Gal4, LexA-VP16
- the determination of the binding region with the help of the two hybrid system is based on the same considerations as for the / n-wrro reconstitution of the binding: If the change or deletion of an amino acid or sequence leads to the loss of binding activity, it is part of the interaction-dependent sequence, and vice versa , if the modification or deletion of a sequence does not lead to the loss of binding activity, it is not part of the interaction-dependent sequence.
- the interaction-related sequence can be defined by examining a large number of fragments of the protein with regard to their interaction and determining which parts of the protein are always present in the interacting or interacting fragments. This area that is always present is the interaction-dependent sequence.
- individual amino acids can be changed by targeted mutations. Loss or increase in binding activity indicates that these positions are directly involved in the interaction.
- the complexes which are particularly preferred for in vitro applications include the thermostable complexes of prokaryotic and eukaryotic replication apparatuses, which often contain polymerases as an important enzyme activity.
- the replication apparatus includes a variety of components. These include, inter alia, a) proteins having polymerase activity, b) proteins which are involved in the formation of a clamp structure, the clamp structure having, inter alia, the task of binding polymerase activity to its template or template and stabilizing the binding and thus to change the dissociation constant of the complex of polymerase and nucleic acid accordingly, c) proteins which load the clip onto the template, d) proteins which stabilize the template and, if appropriate, e) proteins which carry the polymerase to the template.
- Proteins having polymerase activity are understood here to mean in particular those proteins which are able to bind one or more nucleotides or nucleosides to a nucleotide or nucleoside or polynucleotide or polynucleoside. In each of the above cases, these can be ribunucleotides / ribunucleosides or deoxynucleotides / deoxynucleosides or polymers thereof. In addition to DNA polymerases, these proteins also include RNA polymerases, regardless of whether a template is required for the polymerization reaction of the protein or not. Proteins having this polymerase activity are thus also proteins having nucleic acid synthesis activity.
- Elongation protein is also to be understood here to mean a protein or complex which has polymerase activity and which has at least one or more of the following properties: use of RNA as a template, use of DNA as a template, synthesis of RNA, synthesis of DNA, exonuclease activity in 5'-3 'direction or exonuclease activity in 3'-5' direction, strand displacement activity and processivity or non-processivity.
- DNA polymerases belong to a group of enzymes that use single-stranded DNA as a template for the synthesis of a complementary DNA strand. These enzymes play an important role in nucleic acid metabolism, including the processes of DNA replication, repair and recombination. DNA polymerases have been identified in all cellular organisms, from bacterial to human cells, in many viruses and in bacteriophages (Kornberg, A. & Baker, TA (1991) DNA replication WH Freeman, New York, NY).
- the archaebacteria and the eubacteria are grouped together to form the group of procaryonts, the organisms without a real cell nucleus, and the eukaryotes, the organisms with a real cell nucleus, are compared.
- common to many polymerases from a wide variety of organisms are often similarities in the amino acid sequence and similarities in structure (Wang, J., Sattar, AKMA; Wang, CC, Karam, JD, Königsberg, WH & Steitz, TA (1997) Crystal Structure of pol ⁇ familily replication DNA polymerase from bacteriophage RB69.Cell 89, 1087-1099).
- Organisms like humans have a large number of DNA-dependent polymerases, but not all of them are responsible for DNA replication, but some also carry out DNA repair.
- replicative DNA polymerases mostly consist of protein complexes with multiple units that replicate the chromosomes of cellular organisms and viruses.
- a general property of these replicating polymerases is generally high processivity, that is, their ability to polymerize thousands of nucleotides without deviating from the DNA template. dissociate (Kornberg, A. & Baker, TA (1991) DNA replication. WH Freeman, New York, NY).
- DNA polymerases are characterized, among other things, by two properties, their elongation rate, that is the number of nucleotides that they can incorporate into a growing DNA strand per second and their dissociation constant. If the polymerase dissociates from the strand again after each nucleotide incorporation step into the growing chain (i.e. one elongation step occurs per binding event), then the processivity has the value 1 and the polymerase is not processive.
- the polymerase remains attached to the strand for repeated nucleic acid incorporations, then the elongation or the replication mode and thus also the polymerase is referred to as processive and can reach a value of several thousand (see also: Methods in Enzymology Volume 262, DNA Replication, Edited by JL Campbell, Academic press 1995, pp. 270-280).
- the proteins mentioned under b) form structures that are either open or closed, for example ring-shaped or semi-ring-shaped structures. Such structures can be formed by one or more species of proteins. It is possible that one of said protein species has polymerase activity.
- glide clip proteins The proteins responsible for the formation of these structures, if they have no polymerase activity, are hereinafter referred to as “glide clip proteins” or “clip proteins”.
- the replication apparatus in Archaea is known to be similar to the eukaryotic replication apparatus, although the genome organization in eukaryotes and Archaea is completely different and the cellular structure of the eubacteria is similar to that of the Archaea. (Edgell, DR and Doolittle, WF (1997). Archaea and the origin (s) of DNA replication proteins. Cell 89, 995-998.).
- the glide clip is often linked to an elongation protein via one or more other proteins, in other words coupled to the elongation protein.
- Such a coupling protein is hereinafter referred to as a coupling protein, where the coupling can optionally be carried out via a plurality of coupling proteins.
- the glide clip cannot position itself around the DNA in vivo, but must be fixed around the DNA.
- a protein complex consists of a large number of subunits.
- the protein complex recognizes the 3 'end of the primer of a "phmer template duplex", which is to be supplemented to a longer double strand by incorporation of nucleotides, and positions the glide clip around the DNA.
- the phage expresses its own catalytic polymerase, the T7 polymerase, which binds to the gene product of gene 5 and a protein from the host Esche ⁇ chia coli, the thioredoxin, and which, as a replicase, enables highly processing DNA replication.
- a bracket is also formed here, but this bracket does not have the same structure as, for example, in the case of eukaryotic PCNA.
- a processivity factor is preferably a compound or molecule that affects the processivity of a polymerase, preferably increases.
- the sliding clamp proteins represent an example of a processivity factor.
- this coupling protein is the small subunit of the ⁇ polymerase (Zhang, S.-J., Zeng, X.-R., Zhang, P., Toomey, NL, Chuang, RY, Chang, L.-S., and Lee, MYWT (1994.
- thermostable DNA polymerases in the polymerase chain reaction (PCR).
- DNA is newly synthesized using primers, templates (also called templates), nucleotides, a DNA polymerase, a corresponding buffer and under suitable reaction conditions.
- a thermostable polymerase is preferably used which survives the cyclic thermal melting of the DNA strands. So Taq DNA polymerase is often used (US Patent 4,965,188).
- the processivity of the Taq DNA polymerase is, as stated above, relatively low compared to that of the T7 polymerase.
- DNA polymerases are also used in DNA sequence determination (Sanger et al., Proc. N ⁇ tl. Acad. Sei., USA 74: 5463-5467 (1997)).
- One of the polymerases used here can be, for example, the above-mentioned tag polymerase (US Pat. No. 5,075, 216) or the polymerase from Thermotoga neapolitana (WO 96/10640) or other thermostable polymerases. Newer methods couple the exponential amplification and the sequencing of a DNA fragment in one step, so that it is possible to sequence genomic DNA directly.
- DEXAS Direct exponential amplification and sequencing
- a further development of this method consists in the use of a polymerase mixture, one of the two polymerase discriminating between ddNTPs and dNTPs, while the second has a reduced ability to discriminate (Nucleic Acids Res 1997 May 15; 25 (10): 2032-2034 Direct DNA sequence determination from total genomic DNA.Kilger C, Pääbo S).
- DNA polymerases are also used in the reverse transcription of RNA into DNA.
- RNA serves as a template and the polymerase synthesizes a complementary DNA strand.
- thermostable DNA polymerase from the organism Thermus thermusphilus (Tth) (U.S. Patent 5,322,770).
- Some polymerases have a proof-reading activity, ie a 3'-5 'exonuclease activity. This property is particularly desirable if that is synthesizing product to be manufactured with a low error rate in nucleotide incorporation.
- Polymerases from the organism Pyrococcus wosei are an example.
- the above-mentioned elongation proteins which are used in the above-mentioned applications, mostly do not belong to the actual replication enzymes in vivo, but are mostly enzymes that are believed to be involved in DNA repair, which is why their processivity is relative is low.
- every organism has a large number of polymerases which have a large number of properties.
- Such elongation proteins as stated above, e.g. the following properties: use of RNA as a template, use of DNA as a template, synthesis of RNA, synthesis of DNA, exonuclease activity in 5'-3 'direction and exonuclease activity in 3'-5' direction, strand displacement activity , Processivity or non-processivity or thermostability or thermosensitivity.
- a protein complex combines one or more of these properties.
- replication complexes are often present in vivo, the processivity of which, as explained above, is increased by the presence of a glide clip protein.
- DNA-modifying activity is understood here to mean any enzymatic activity which leads to a chemical, physical or structural change in a starting nucleic acid. It also happens that DNA-modifying activity only comes about through interaction with at least one other protein comes. It is also possible that a DNA-modifying activity is reduced or increased by the interaction with at least one further protein.
- a protein complex often arises in vivo which, for example, bears the sum of the individual activities or whose activity is improved compared to the individual activity.
- in vivo complexes comprising nucleic acid synthesis activity often have further ones for the technical, i.e. in vitro application desired properties that go beyond the actual nucleic acid synthesis activity and are contributed by the other components forming the complex.
- a direct technical use of such in vivo complexes z For example, for purposes of sequencing DNA, performing a polymer chain reaction, or introducing labels into nucleic acids has so far failed for a number of reasons. One reason was and is the lack of knowledge of all factors or individual components involved in the formation of the complex of interest. Another reason is that the multi-component complex has one or more undesirable properties in addition to those desired.
- the present invention is therefore based on the object of providing proteins with a nucleic acid synthesis activity which have an increased processivity. It is a further object of the present invention to provide a method which allows the construction of such proteins.
- a recombinant chimeric protein comprising
- the interaction mediating sequence forms a complex of nucleic acid synthesis activity and glide clip protein and the complex is different from the complex which the nucleic acid synthesis activity and / or the glide clip protein forms with its natural interaction partner (s).
- the object is achieved by a chimeric protein comprising a portion having nucleic acid synthesis activity, the portion coming from a portion having a nucleic acid synthesis activity and an interaction-imparting portion, and at least one interaction-imparting portion, the interaction-imparting portion
- the proportion is different from the interaction-mediating proportion of the base protein.
- the protein comprises the interaction-mediating portion of the base protein.
- the object is achieved by a chimeric protein comprising a portion having nucleic acid synthesis activity, the portion consisting of is derived from a base protein which comprises a part which has a nucleic acid synthesis activity but no part which mediates interaction, and an part which mediates interaction.
- the interaction-mediating portion mediates a bond between the nucleic acid synthesis activity and a factor influencing the synthesis performance of the nucleic acid synthesis activity.
- the factor is a glide clip protein.
- the nucleic acid synthesis activity comprises a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ ID NO .: 1, 2 and 3.
- the glide clip protein comprises a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ ID NO .: 4, 5, 6 and 7.
- the interaction-mediating sequence comprises a consensus peptide sequence which is selected from the group comprising the sequences SEQ ID NO .: 8, 9, 10 11 and 12. In a particularly preferred embodiment, it is provided that the interaction-mediating sequence comprises a consensus peptide sequence according to SEQ ID NO .: 8.
- the interaction-mediating sequence is at the C-terminal end of the sequence carrying the nucleic acid synthesis activity.
- a linker is arranged between the interaction mediating sequence and the sequence carrying the nucleic acid activity.
- the recombinant chimeric protein can be thermostable.
- the protein has a DNA polymerase activity. It is particularly preferred if the proteins have a 3 ' -5 ' exonuclease activity.
- the protein has an RNA polymerase activity.
- the protein has a reverse transcriptase activity.
- the incorporation rate of dNTPs and ddNTPs differs by a factor of less than 5 due to the nucleic acid synthesis activity.
- the object is achieved by a complex comprising
- the glide clip protein comprises a consensus peptide sequence which is selected from the group comprising the sequences according to SEQ ID NO .: 4, 5, 6 and 7.
- the complex further comprises a nucleic acid.
- the object is achieved by a nucleic acid which codes for a chimeric protein according to the invention, in particular recombinant protein.
- the object is achieved by a vector comprising the nucleic acid according to the invention.
- the vector is an expression vector.
- the object is achieved by a cell which comprises the vector according to the invention.
- the object is further achieved by using the chimeric protein according to the invention for the elongation of nucleic acids.
- the object is further achieved by using the chimeric protein according to the invention for the amplification of nucleic acids.
- the object is further achieved by the use of the chimeric protein according to the invention for the reverse transcription of RNA in DNA.
- the object is further achieved by using the chimeric protein according to the invention for sequencing nucleic acids, in particular DNA.
- the object is achieved by a kit, in particular a reagent kit for elongation and / or amplification and / or reverse trans description and / or sequencing and / or labeling of nucleic acids, which comprises in one or more separate containers: a) an inventive, preferably recombinant chimeric and / or b) an inventive complex and c) preferably optionally at least one primer, buffer, nucleotide, Cofactors and / or pyrophosphatase.
- a kit in particular a reagent kit for elongation and / or amplification and / or reverse trans description and / or sequencing and / or labeling of nucleic acids, which comprises in one or more separate containers: a) an inventive, preferably recombinant chimeric and / or b) an inventive complex and c) preferably optionally at least one primer, buffer, nucleotide, Cofactors and / or pyrophosphatase.
- kits it is provided that, in addition to substances a) and / or b), it comprises deoxynucleotides or / and derivatives thereof for the amplification of nucleic acids.
- the kit comprises a DNA polymerase with 3'-5 'exonuclease activity.
- the kit for reverse transcription contains substances according to a) and / or b) which have reverse transcriptase activity, and preferably deoxynucleotides and / or derivatives thereof.
- kit contains, in addition to deoxynucleotides or ribonucleotides or / and their derivatives, dideoxynucleotides or / and their derivatives for sequencing.
- the object is achieved by a method for template-dependent elongation of nucleic acids, the nucleic acid to be elongated or at least a strand thereof being provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a part of or a flanking region of the nucleic acid to be elongated and a primer elongation is carried out by a polymerase in the presence of nucleotides, it being provided that a chimeric, preferably chimeric protein according to the invention is used as the polymerase is used and that preferably a slide clamp protein is present in the reaction.
- the object is achieved by a method for amplifying a nucleic acid, it being provided that the nucleic acid to be amplified is crosslinked with at least two primers under hybridization conditions, each of the two primers in each case complementary to a part of or a flanking one
- the region of the nucleic acid to be amplified is and a primer elongation is carried out by a polymerase in the presence of nucleotides.
- a chimeric protein according to the invention, in particular a recombinant protein, is used as the polymerase and a slide clamp protein is preferably added to the reaction.
- a polymerase chain reaction is carried out.
- the reaction mixture comprises two DNA polymerases, at least one of which has a 3'-5 'exonuclease activity, the 3 ' -5 ' exonuclease activity being either by the chimeric protein or by a further polymerase Reaction mixture is added.
- two chimeric proteins, in particular recombinant one of which is one that has DNA polymerase activity and the other is one that has 3 ' -5 ' exonuclease activity having.
- the nucleic acid adjacent area is complementary, a template-dependent Elongation or reverse transcription is carried out using deoxynucleotides and dideoxynucleotides or their respective derivatives according to the Sanger method.
- At least one marker is inserted when the nucleic acids are elongated.
- an agent which is selected from the group comprising labeled primers, labeled deoxynucleotides and derivatives thereof, labeled dideoxynucleotides and derivatives thereof and labeled ribonucleotides and derivatives thereof.
- the object is achieved by a method for labeling nucleic acids by generating individual breaks in phosphodiester linkages of the nucleic acid chain and replacing a nucleotide at the break points with a labeled nucleotide with the aid of a polymerase, it being provided that a polymerase chimeric protein according to the invention, in particular recombinant chimeric protein is used.
- the object is achieved by a method for producing a chimeric protein which comprises a base sequence and a heterologous interaction-dependent sequence and, as a result of the interaction-dependent sequence, forms a bond with an interaction partner or such a bond is strengthened, whereby
- the sequence of the donor protein or acceptor protein which determines the interaction between the two interaction partners is determined from an interaction system comprising a protein referred to as donor protein and a protein as acceptor protein; and b) the interaction-causing sequence is introduced into a receiving protein which comprises the base sequence and which is different from the donor protein and the acceptor protein.
- the donor protein and the acceptor protein form a complex that binds nucleic acid.
- the donor protein and the acceptor protein form a complex which has an activity which is selected from the group, the polymerase activity, DNA binding activity, RNA binding activity, 5 ' -3 ' exonuclease activity, 3 ' -5 ' exonuclease activity and ligase activity.
- the donor protein is selected from the group comprising elongation protein, glide clip proteins, glide clip loader protein and coupling proteins.
- the acceptor protein is selected from the group comprising elongation protein, glide clip proteins, glide clip loader protein and coupling proteins.
- the receiving protein is selected from the group comprising elongation protein, glide clip proteins, glide clip loader protein and coupling proteins.
- step a) is repeated several times and a consensus sequence which represents an interaction-dependent sequence is determined from the thus determined interaction-dependent sequences, and in step b) as an interaction-dependent sequence in one of the donor protein and the acceptor protein various receiving protein comprising a base sequence is introduced.
- the object is achieved by a chimeric protein which can be obtained by the process according to the invention.
- the base sequence is part of the amino acid sequence of a protein selected from the group consisting of elongation proteins, glide clip proteins, glide clip protein and coupling proteins.
- an in vitro complex for template-dependent elongation of nucleic acid comprising a slide clip protein and an elongation protein, at least one of the proteins being a chimeric protein according to the invention.
- An embodiment in which the complex is thermostable is particularly preferred.
- each of the chimeric proteins according to the invention can be a recombinant chimeric protein.
- the object is also achieved by recombinant chimeric protein comprising a) a first domain with nucleic acid synthesis activity, and b) an interaction-mediating sequence, characterized in that the interaction-mediating sequence forms a complex of nucleic acid synthesis activity and gliding clip protein, the complex being different of the complex that the nucleic acid synthesis activity and / or the glide clip protein forms with their natural interaction partner (s).
- nucleic acid synthesis activity to a glide clip protein which has the nucleic acid synthesis activity as it occurs in nature, for example. does not come can bind.
- the natural interaction partner is therefore a partner as it can be present within an organism under normal physiological conditions to which the nucleic acid synthesis activity is bound.
- the object is achieved by a chimeric protein comprising a portion having nucleic acid synthesis activity, the portion coming from a portion having a nucleic acid synthesis activity and an interaction-imparting portion, and at least one interaction-imparting portion, the interaction-imparting portion being different from that Interaction-mediating portion of the base protein, and a chimeric protein comprising a portion having nucleic acid synthesis activity, the portion originating from a base protein which comprises a portion having nucleic acid synthesis activity but no interaction-imparting portion, and an interaction-imparting portion.
- the object is further achieved by a method for template-dependent elongation of nucleic acids, the nucleic acid to be elongated or at least one strand thereof being provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a part of or a flanking region of the is to be elongated nucleic acid and a primer elongation is carried out by a polymerase in the presence of nucleotides, characterized in that a recombinant chimeric protein according to the invention is used as the polymerase and that preferably a slide clamp protein is present in the reaction.
- “recombinant” is to be understood when the chimeric protein is produced, for example, by genetic engineering (see “Genetic Technology”, Römpp Basislexikon Chemie, Georg Thieme Verlag 1998) or, for example, when it is chemically synthesized.
- the invention is based on the surprising finding that it is possible, starting from a protein having a nucleic acid synthesis activity, hereinafter referred to as the basic protein, to increase the processivity of this protein, more precisely the nucleic acid synthesis activity, by combining it with a factor which increases the processivity, such as a Sliding clamp protein interacts with which the basic protein as such cannot interact, or in the event that it can interact, this is not associated with an increase in processivity.
- a factor which increases the processivity such as a Sliding clamp protein interacts with which the basic protein as such cannot interact, or in the event that it can interact, this is not associated with an increase in processivity.
- nucleic acid synthesis activity-bearing protein with a group of several amino acids, typically in the form of a consecutive amino acid sequence, referred to herein as an interaction-mediating or interaction-causing sequence, and thus an interaction between the said Protein and a factor that increases processivity is only possible.
- This overcomes the above-described problem of the prior art, consisting in a lack of compatibility of the actually desired individual components of a complex carrying a nucleic acid synthesis activity.
- the basic protein on which the chimeric protein is based can be in two basic forms.
- the base protein comprises only the nucleic acid synthesis activity, but not a sequence (also referred to herein as a domain, which, particularly in vivo, can or could mediate an interaction between the nucleic acid synthesis activity and a processivity factor, in particular not such that the interaction causes a Increased processivity is achieved.
- the base protein comprises a nucleic acid synthesis activity and additionally a sequence which, in particular in vivo, can or could mediate an interaction between the nucleic acid synthesis activity and a processivity factor, in particular in such a way that an increase in the processivity is achieved by the interaction.
- the chimeric protein according to the invention can be present in various basic embodiments based on the different forms of the basic protein.
- a first embodiment of the chimeric protein provides that the first form of the base protein is used and a sequence mediating an interaction with a factor which increases the processivity of the nucleic acid synthesis activity is added to it. This addition is typically made in that the interaction-mediating sequence follows the sequence of the nucleic acid synthesis activity, optionally separated by a linker. In this first embodiment of the chimeric protein, the base protein is thus supplemented by an interaction mediating sequence.
- the interaction inherent in the basic protein is exchanged with a sequence mediating the factor increasing the processivity of the nucleic acid synthesis activity by another sequence mediating an interaction with a factor increasing the processivity of the nucleic acid synthesis activity.
- This other sequence can originate from another gene of the same organism, from the same gene from another organism or from a different gene from another organism and is therefore in any case of a different origin.
- such a construction possibly for the first time, but possibly only to an increased extent, enables an interaction between the nucleic acid synthesis activity of the base protein and a factor which increases the processivity of the nucleic acid synthesis activity.
- the second form of the basic protein is used.
- the sequence inherent in the basic protein is an interaction with a sequence mediating the factor increasing the processivity of the nucleic acid synthesis activity by another sequence mediating an interaction with a factor increasing the processivity of the nucleic acid synthesis activity.
- This other sequence can originate from another gene of the same organism, from the same gene from another organism or from a different gene from another organism and is therefore in any case of a different origin.
- the (amino acid) sequence of such a chimeric protein is typically different from the sequence of the base protein. Different. A further, but not obligatory consequence can be seen in the fact that the complex formed by the chimeric protein of the protein and processivity-increasing factor having nucleic acid synthesis activity is different from the complex of base protein and processivity-increasing factor.
- Suitable as basic proteins are all proteins, polymerases and elongation proteins mentioned in the introduction, the disclosure of which is hereby incorporated by reference. The same applies to the other components, in particular those of the replication apparatus, such as processivity factors, glide clip proteins and interaction-mediating or interaction-dependent sequences. Finally, the definitions given in the introduction also apply to this part of the disclosure.
- nucleic acid synthesis activity can originate from a large number of organisms, it is preferred if it originates, for example, from the organism Carboxydotherm hydrogenoformans (European Patent Application EP 0 834 569 A1) or one of the organisms such as Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruher, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sp.
- Carboxydotherm hydrogenoformans European Patent Application EP 0 834 569 A1
- the organisms such as Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruher, Thermus scotoductus, Thermus silvanus
- Thermus thermusphilus Therotoga maritima, Therotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, or Bacillus stearothermophilus.
- nucleic acid sequences are usually addressed directly as nucleic acid sequences.
- nucleic acids coding for the recombinant chimeric proteins according to the invention can easily be determined by those skilled in the art using the genetic code and then synthesized.
- Suitable vectors for cloning and expression of the recombinant chimeric proteins according to the invention and methods for their preferred recombinant production are also known to the experts (see, for example, Maniatis et al .; loc. Cit.).
- the recombinant chimeric protein according to the invention can be used to elongate nucleic acids, e.g. B. for polymerase chain reaction, DNA sequencing, for labeling nucleic acids and other reactions that involve the in vitro synthesis of nucleic acids.
- Another object of the present invention is therefore a method for template-dependent elongation, wherein the nucleic acid to be elongated or at least a strand thereof is provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a part of or a flanking region of the is elongating nucleic acid and a primer elongation is carried out by a polymerase in the presence of nucleotides, a recombinant chimeric protein according to the invention being used as the polymerase and, in a preferred embodiment, a slide clamp protein also being present in the reaction or in the reaction mixture.
- Methods for the template-dependent elongation of nucleic acids in which the elongation takes place starting from a primer which has been hybridized to the template nucleic acid and provides a free 3'-OH end for the elongation, are known to the person skilled in the art.
- a polymerase chain reaction is carried out for the amplification. This is usually based on a double-stranded DNA sequence from which a specific target area is to be amplified.
- two primers are used, which are complementary to the regions flanking the target sequence in each case on a partial strand of the DNA double strand.
- the DNA double strands are first denatured, in particular thermally melted.
- thermostable in vitro complex itself has reverse transcriptase activity.
- a recombinant chimeric protein according to the invention is also used for the reverse transcription of RNA into DNA preferred according to the invention, the nucleic acid synthesis activity itself having a reverse transcriptase activity.
- This reverse transcriptase activity can be the only polymerase activity, but can also be present in addition to an existing 5'-3 'DNA polymerase activity.
- a preferred embodiment of the recombinant chimeric protein according to the invention comprises the elongation protein from the organism Carboxydothermus hydrogenoformans as disclosed in EP-A 0 834 569.
- a further preferred use of the recombinant chimeric protein according to the invention is the sequencing of nucleic acids starting from at least one primer which is sufficiently complementary to a part of the nucleic acid to be sequenced, again using template-dependent elongation or, if RNA is sequenced, using reverse transcription of deoxynucleotides and dideoxynucleotides according to the Sanger method.
- the respective derivatives described above are also considered suitable as deoxynucleotides or dideoxynucleotides.
- it is preferred for the methods according to the invention for elongating nucleic acids that the nucleic acids formed are labeled.
- the present invention furthermore relates to a method for labeling nucleic acids by inserting individual breaks in phosphodiester bonds in the nucleic acid chain and replacing a nucleotide at the break points with a labeled nucleotide using a polymerase, a thermostable in vitro complex according to the invention being used as the polymerase.
- nick translation enables simple labeling of nucleic acids. All of the labeled ribonucleotides or deoxyribonucleotides or derivatives thereof already described above are suitable for this as long as the polymerase accepts them as a substrate.
- Fig. 2 is a schematic representation of the recombinant according to the invention
- FIG. 6A shows the entire sequence of an embodiment of a recombinant chimeric protein according to the invention
- 6B is a graphic representation of the basic structure of a chimeric protein according to the invention.
- FIG. 7 shows the result of a polymerase chain reaction carried out using a chimeric protein according to the invention
- 8A shows all fragments of Af0497 which interact with the slide clip protein of Archaeoglobus fulgidus (Af0335);
- 8B shows an alignment of C-terminal sequences of different genes from nArchaeglobus fulgidus;
- FIG. 10 shows the result of the amplification of genomic DNA using a recombinant chimeric protein according to the invention.
- Example 1 Application of the yeast two-hybrid system for determining the amino acids which are important for the interaction between replication factors and gliding clip protein
- GKP slide clamp proteins
- RF replication factors
- the open reading frames of the genes are then amplified by PCR from Archaeoglobus fulgidus genomic DNA.
- the DNA thus obtained is cloned into the vectors pGBT9 and pGAD424.
- Other vectors that are used in the two-hybrid system are also suitable for the method described below (these include, for example, pAD-GAL4-2.1, pBD-GAL4, pBD-GAL4 Cam, pCMV-AD, pCMV-BD, pMyr, pSos, pACT2, pAS2-1, pHISi, pLexA, pM, pHISi-1, pB42AD, pVP16, pGAD10, pGBKT7, pLacZi, p ⁇ op-lacZ, pGAD GH, pGilda, pAD GL, pGADT7, pGBDU, pDBLeu, pPC86, pDB86 die
- modified clones are cloned into the same vectors.
- the modified clones are deletion mutations and mutations relating to one or more amino acids of the GKP and / or the RF .
- the ability of the modified clones to interact with one another in the two-hybrid system is then measured. This makes it possible to determine domains or even individual amino acid residues that are important or essential for the interaction.
- deletions There are a number of ways to insert deletions into a gene.
- One method of creating deletions is to make DNA fragments that contain the genes for GKP or RF. These fragments are obtained from a suitable vector either by PCR or by restriction digestion. Genomic DNA from the organism from which the two proteins originate is also used. These various DNA fragments and the genomic DNA are crushed by ultrasound treatment and fragments that have lengths between the total length of the genes and about 100 bases are purified by preparative agarose gel electrophresis. The ends of the fragments are filled in or digested by treatment with a suitable enzyme (Klenow, Pwo polymerase, or others), so that both strands have the same length and are therefore blunt.
- a suitable enzyme Klenow, Pwo polymerase, or others
- fragments are now inserted by ligation into a Smal cut (or otherwise linearized and blunted) pGAD424 and pGBT9 vector, or other suitable vectors.
- the DNA of these banks is grown and purified after transformation in Escherichia coli.
- the two banks are transformed into a haploid yeast strain suitable for the two hybrid system, so that the fusions from GKP or RF with the GAL4 DNA-binding domain are in a strain with a different mating type than the fusions from RF or GKP of GAL4 activation domain.
- diploid cells are now generated in which the plasmids from both banks are present in the same cell.
- PJ69-4 James P, Halladay J, Craig EA, Genetics 1996 Dec; 144 (4): 1425-36
- any other strain with a genotype suitable for the two-hybrid system.
- Cells in which the reporter genes are activated are isolated, and the plasmid DNA is obtained therefrom by preparation, or the inserts are specifically amplified by PCR.
- the sequences of the fusion fragments are determined by DNA sequence analysis.
- the binding region (or regions) is (are) determined by determining those regions which are found in all clones (or which are always found in certain groups of clones) where the two fusion proteins comprising RF and GKP interact. This determination is carried out four times for each of the genes: once with the bank in the vector with the activation domain (preferentially: pGAD424) and once with the bank in the vector with the DNA binding domain (preferentially: pGTB9), in each case once with the total length clone of the binding partner and once with the gene bank of fragments.
- Example 2 Other methods of mapping the binding region of GKP and RF
- GKP and RF were obtained as recombinant proteins.
- One of the two proteins was immobilized on a carrier, then the other protein was added in unbound form to allow binding to the immobilized partner. Free, i.e. unbound material was washed out and the bound protein e.g. eluted by denaturation. The amount of protein bound is a measure of binding (e.g. used in Anderson D, Koch CA, Gray L, Ellis C, Moran MF, Pawson T, Science 1990 Nov 16; 250 (4983): 979-82).
- deletion mutants of the proteins and of mutants with an altered amino acid sequence allowed the mapping of the binding region.
- Deletions can be generated by proteolytic digestion or by means of recombinant expression of deleted genes.
- the binding region can be determined by co-immunoprecipitation from cell extracts which contain deleted forms of the proteins.
- the competition of binding by peptides can provide information about the sequence of the binding region.
- Another way to map the binding region is to produce peptides and measure the binding of the peptides to the other protein.
- the sequence of the peptides can be random (Songyang Z, Prog Biophys Mol Biol 1999; 71 (3-4): 359-72) or based on the amino acid sequence of the protein whose binding region is to be identified (petide scans, Brix J, Rudiger S, Bukau B, Schneider-Mergener J, Pfanner N, J Biol Chem 1999 Jun 4; 274 (23): 16522-30).
- Such peptides can be made by chemical synthesis or other methods such as phage display and offered for binding. An example is shown in FIG. 3. Alignments were used to determine various consensus sequences that are suitable for the production of such peptides.
- Another method is to produce antibodies against epitopes of the protein whose binding region is to be identified. If the antibody inhibits binding, the epitope of the antibody overlaps with the binding region (e.g. Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA, Nature 1994 Apr 28; 368 (6474): 871-4).
- the binding region e.g. Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA, Nature 1994 Apr 28; 368 (6474): 871-4.
- one way of determining or mapping the binding region is to clarify the fine structure of the complex using the methods of X-ray structure analysis, nuclear magnetic resonance or electron microscopy.
- Example 3 Study of the interaction of proteins from Archaeoglobus fulgidus using the yeast two-hybrid system (Y2H)
- a positive control was amplified by PCR, cloned into the vectors pGBT9 and pGAD424 (see also horizontal rows in FIG.
- those cells which carry both vectors and in which the expression products of these two vectors also bind to one another grow in a histidine and adenine deficiency medium.
- the interaction of the fusion proteins causes the reconstitution of a functional transcription factor that initiates the transcription of the reporter genes.
- the production of the proteins encoded on the reporter genes leads to the abolition of the histidine and adenine auxotrophy. This is due to the fact that, as a result of the binding of the expression products, a transcription is initiated which leads to the cells being able to grow.
- the DNA of the gene for Af0497 was first obtained by restriction of a suitable vector which contained an elongation protein which comprised an interaction-dependent sequence , This DNA was then ultrasonically fragmented and ligated into the two vectors pGAD424 and pGBDU. This resulted in banks of different fragments of the gene in the two vectors. Next, using the yeast two hybrid system, it was determined which of these fragments could interact with Af0335. For this purpose, both banks were transformed into suitable yeast strains (pGAD424: PJ69-4a, PGBDU: PJ69-4alpha).
- the polymerase Taq does not bind to the GKP (PCNA from Archaeoglobus fulgidus), and no interaction could be measured in the yeast two-hybrid system.
- GKP PCNA from Archaeoglobus fulgidus
- the grafting of the interaction-causing sequence of Af0497 to Taq caused a specific interaction of Taq with the gliding clip protein Af0335.
- the results of the corresponding Y2H expehments are shown in FIG. 4.
- this fragment (the carboxy-terminal end of the elongation protein Af0497) to cause an interaction with the gliding clip protein Af0335 can be transplanted or transferred to another protein, in particular to another polymerase, in order to interact there with the actually produce specific gliding clip protein for Af0497.
- Such interaction-related sequences are contained in the last 50 amino acids of the Af0497 protein. Examination of the proteins that interact with PCNA revealed that they all contain a motif that is located just before the carboxy-terminal end of the amino acid sequence. A listing of the related sequences is shown in Fig. 8B, with a black bar indicating the conserved area.
- This example shows the influence of PCNA on the efficiency of a PCR reaction.
- a 463 bp fragment was amplified from plasmid DNA.
- the taq fusion protein as also shown in FIG. 6A, was used as the polymerase.
- the reaction conditions for the PCR were as follows: 0.4 mM of each dNTP (pH 8.3) and 20 pmol of each primer in one reaction.
- a first primer (SEQ ID NO .: 13) with the sequence 5'-AGGGCGTGGTGCGGAGGGCGGT-3 'and a second primer (SEQ ID NO .: 14) with the sequence 5'-TCGAGCGGCCGCCCGGGCAGGT-3' were used.
- Example 6 Yeast two-hybrid system for the detection of the interaction between the elongation protein Af 0497 from Archaeoglobus fulgidus and the slide clamp protein Af 0335 from Archaeoglobus fulgidus
- Fig. 9 shows the results of a Y2H experiment where row A is populated with cells carrying the empty pGAD424 vector (Clontech, Palo Alto, USA) so that a transcription activation domain is expressed; the row B with cells which carry the pGAD424 vector from which the Sacharomyces cerevesiae gene CDC48 is expressed as a fusion protein with the transcription activation domain; row C is populated with cells carrying the pGAD424 vector, from which the Archaeoglobus fulgidus glide gene is expressed as a fusion protein with the transcriptional activation domain; row D is not populated with cells and row E is populated with cells that carry the pGAD424 vector from which the elongation protein gene from Archaeoglobus fulgidus is expressed as a fusion protein with the transcription activation domain.
- row A is populated with cells carrying the empty pGAD424 vector (Clontech, Palo Alto, USA) so that a transcription activ
- Column 1 is populated with cells that carry the empty pGBT9 vector (Clontech, Palo Alto, USA); column 2 is populated with cells which carry the pGBT9 vector, of which the Saccharomyces cerevisiae gene UFD3 is expressed as a fusion protein with the DNA binding domain; column 3 is populated with cells which carry the pGBT9 vector, from which the sliding clip protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain; column 4 is populated with cells that carry the pGBT9 vector from which the coupling protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain, and column 5 is populated with cells that carry the pGBT9 vector from which the elongation protein originates Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain.
- Example 7 Polymerase chain reaction of genomic DNA using a chimeric elongation protein
- Example 7 shows the influence of the slide clamp protein on the efficiency of a PCR reaction on longer DNA fragments using the recombinant recombinant chimeric protein according to the invention.
- a 4954bp fragment was amplified from human genomic DNA.
- the recombinant chimeric according to the invention was used as polymerase Protein used.
- the conditions correspond to the standard conditions of a PCR reaction: pH 8.3, 0.4 mM of each dNTP and 20 pmol of each primer in one reaction.
- a first primer ((SEQ ID NO .: 15): 5'-AGGAACAACATATGACGCACTCT-3 ')
- a second primer ((SEQ ID NO . : 16): (5'-TAGGTGGCCTGCAGTAATGTTAG-3') were used.
- FIG. 1 shows sequence alignments of a total of four different elongation protein domains from different organisms, for example for the elongation protein 1 from humans, Archaeglobus fulgidus, Methanococcus thermoautotrophicusm, PHBT (Pyrococcus horikoshii), and Methanococcus janashii.
- the figures often have a consensus sequence in a spelling, in which varying positions are indicated by a single character.
- the square ones Amino acids given in brackets represent those amino acids, expressed in the one-letter code, that can appear at the specific position.
- amino acids are named in accordance with the standard IUPAC one-letter nomenclature and listed in accordance with the Prosite Pattern description standard.
- amino acid groups are often summarized:
- Figure 1 gives four different consensus sequences for different areas of elongation proteins, e.g. the ionization protein 3 can often be found in eubacteria, the elongation protein 4 also includes the Taq polymerase and can often be found in Pol I type polymerases. Elongation protein 3 thus shows an alignment of a conserved region of the elongation protein from eubacteria, and the consensus sequences derived therefrom.
- DP3A_ECOLI DNA Pol III, alpha subunit, Escherichia coli, BB0579: DNA Pol III, alpha subunit, Borrelia burgdorferi, DP3AJHELPY: DNA Pol III, alpha subunit, Helicobacter pylori AA50: Aquifex aeolicus, section 50 and DP3A_ALT DNA Pol III, alpha subunit, Salmonella typhimurium).
- FIG. 2 shows a sketch of a possible form of the recombinant chimeric protein according to the invention, the sliding chamber, as a factor increasing the synthesis activity of the polymerase, binding to the elongation protein carrying the nucleic acid synthesis activity via the domain with an interaction-dependent sequence.
- Fig. 3 shows a sketch of a possible form of the recombinant chimeric protein according to the invention, the sliding chamber, as a factor increasing the synthesis activity of the polymerase, binding to the elongation protein carrying the nucleic acid synthesis activity via the domain with an interaction-dependent sequence.
- Fig. 3 shows a sketch of a possible form of the recombinant chimeric protein according to the invention, the sliding chamber, as a factor increasing the synthesis activity of the polymerase, binding to the elongation protein carrying the nucleic acid synthesis activity via the domain with an interaction-dependent sequence.
- FIG. 3 shows four slide bracket consensus sequences
- region 1 glide clip Human PCNA, as well as an orthologist, from Archaeoglobus fulgidus, from Methanococcus janashii, from Pyrococcus horikoschü and from Methanococcus thermoautothrophicus.
- Region 2 shows an alignment of a second conserved region of the gliding clip from eukaryotes and archae, as well as the consensus sequences derived from them. The alignment was created using a second region of the slide clamps already used in region 1 above.
- Region 3 shows an alignment of a conserved region of the gliding clip from eubacteria, as well as the consensus sequences derived from it.
- the following genes are shown: AAPOL3B, DP3B_ECOLI, S.TYPHIM, DP3B_PROMI, DP3B_PSEPU and DP3B_STRCO (AAPOL3B: Aqufex Aeolicus section 93: DP3B_ECOLI: DNA Pol III, beta chain, Escherichia coli, S.TYPHIM: DNA Polmon typhimurium, P3B_PROMI: DNA Pol III, beta chain, Proteus mirabilis DP3B_PSEPU: DNA Pol III, beta chain, Pseudomonas putida DP3B_STRCO: DNA Pol III, beta chain, Streptomyces coeiicolor).
- Region 4 shows an alignment of a second conserved region of the gliding clip from eubacteria (see organisms in region 3), as well as the consensus sequences derived therefrom
- FIG. 4 shows the interaction of chimeric proteins in the yeast two-hybrid system.
- the result shown in Fig. 4 proves that the property interacts with to condition the gliding clip protein Af0335, can be transplanted to another protein, in particular to another polymerase, in order to produce an interaction with the gliding clip protein.
- the GKP binds itself and the GKP binds a Taqfusionsprotein which comprises an interaction-dependent sequence from Archaeglobus fulgidus.
- Sequence 1 (polymerase gene, Af 0497 homologs): Archaeoglobus fulgidus, Pyrodicti- um occultum, Aeropyrum pernix, Pyrococcus glycovorans, Pyrococcus furiosus, Thermococcus gorgonarius, Pyrococcus abyssi, Pyrococcus horikoshii l
- Sequence 2 (polymerase gene, Af 1722 homologs), Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii and Pyrococcus abyssi and
- Sequence 3 (Af 1347 homologs), Archaeoglobus fulgidus, Pyrococcus abyssi, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and Aeropyrum pernix.
- the interaction-dependent sequences can be introduced into a chimeric protein according to the invention in order to bind a GKP.
- Sequence 4 (RNAse gene, Af 0621 homologs): Archaeoglobus fulgidus, Pyrococcus abyssi, Pyrococcus horikoshii and Arabidopsis thaliana and
- Sequence 5 (polymerase gene): Archaeoglobus fulgidus, Pyrococcus abyssi, Methanococcus thermoautotrophicus, Methanococcus janashii, Mus musculus and Homo sapiens.
- the interaction-dependent sequences can be introduced into a chimeric protein according to the invention in order to bind a GKP.
- 5C shows a list of characters which are used to identify one or more different amino acids, with "amino acid” or the property of the group under “class”, the character used under “key” and the amino acids under “rest” of the group.
- FIG. 6A shows the overall sequence of a preferred embodiment of the recombinant chimeric protein according to the invention.
- the elongation protein and thus the nucleic acid synthesis activity comes from Thermus aquaticus and the interaction-dependent sequence from Archaeglobus fulgidus.
- FIG. 6B shows a graphic overview of the structure of a preferred embodiment of the recombinant chimeric protein according to the invention.
- Elongation protein from Thermus aquaticus.
- interaction-related sequence from Archaeglobus fulgidus.
- FIG. 7 shows the use of a recombinant chimeric protein according to the invention in PCR, the sequence of which is shown in FIG. 6A.
- plasmid DNA pCR 2.1 vector including a 463bp fragment; In Vitrogen, 9704 CH Groningen; Netherlands
- the reactions were applied to the gel in each case with 20 ⁇ l of a 50 ⁇ l mixture, track 7 shows a size standard.
- 8B shows an alignment of the C-terminal sequences of the following genes Af 0497-grUE, Af 1195 Rfc, Af 0264 Rad2-Fen1, Af 0621 RNAseH,, Archaeglobus fulgidus.
- FIG. 9 shows the result of a yeast two-hybrid test and demonstrates a bond between the yeast proteins ufd3 and cdc48 (2B; positive control), between the elongation protein and the glide clip protein (3E; and in the reverse orientation 5C), between the glide clip protein and the glide clip protein ( 3C) and between the coupling protein and the glide clip protein (4C).
- the fusion proteins are expressed from the vectors pGBT9 (vector) in column 1, pGBT9 :: ufd3 (positive control) in 2, pGBT9: .PCNA in 3, pGBT9 :: klUE in 4, pGBT9 :: grUE in 5, pGAD424 (vector) in row A, pGAD424 :: cdc48 in B (positive control), pGAD424 :: PCNA in C, empty in D and pGAD :: grUE in E.
- the amplificate of a PCR reaction based on human genomic DNA with a size of 4954 base pairs is among the following conditions: 95 ° C 5 ' denaturation; 35 x ⁇ 95 ° C 30 " denaturation; 47 ° C 30 " hybridization; 72 ° C 12 ' elongation ⁇ ; 72 ° C 10 additional elongation was carried out, 20 ⁇ l of a 50 ⁇ l batch was applied to the gel.
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DE10082298T DE10082298D2 (de) | 1999-08-06 | 2000-08-07 | Chimäre Proteine |
AU69830/00A AU6983000A (en) | 1999-08-06 | 2000-08-07 | Chimeric proteins |
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EP1456394A2 (de) * | 2001-11-28 | 2004-09-15 | MJ Bioworks Incorporated | Verfahren zur verwendung verbesserter polymerasen |
EP1616033A2 (de) * | 2003-03-25 | 2006-01-18 | Stratagene California | Dna-polymerasefusionen und deren verwendungen |
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US20190055527A1 (en) * | 2015-11-27 | 2019-02-21 | Kyushu University, National University Corporation | Dna polymerase variant |
WO2023059361A1 (en) * | 2021-10-06 | 2023-04-13 | 5Prime Biosciences, Inc. | Polymerases for mixed aqueous-organic media and uses thereof |
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EP1456394A2 (de) * | 2001-11-28 | 2004-09-15 | MJ Bioworks Incorporated | Verfahren zur verwendung verbesserter polymerasen |
US9139873B2 (en) | 2001-11-28 | 2015-09-22 | Bio-Rad Laboratories, Inc. | Methods of using improved polymerases |
US9708598B2 (en) | 2001-11-28 | 2017-07-18 | Bio-Rad Laboratories, Inc. | Methods of using improved polymerases |
EP1616033A2 (de) * | 2003-03-25 | 2006-01-18 | Stratagene California | Dna-polymerasefusionen und deren verwendungen |
EP1616033A4 (de) * | 2003-03-25 | 2007-05-09 | Stratagene California | Dna-polymerasefusionen und deren verwendungen |
US7659100B2 (en) | 2003-03-25 | 2010-02-09 | Stratagene California | DNA polymerase fusions and uses thereof |
US7704712B2 (en) | 2003-03-25 | 2010-04-27 | Stratagene California | DNA polymerase fusions and uses thereof |
EP2194123A1 (de) * | 2003-03-25 | 2010-06-09 | Stratagene California | DNA-Polymerasefusionen und Verwendungen dafür |
US8883454B2 (en) | 2003-03-25 | 2014-11-11 | Agilent Technologies, Inc. | DNA polymerase fusions and uses thereof |
Also Published As
Publication number | Publication date |
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DE19937230A1 (de) | 2001-02-08 |
AU6983000A (en) | 2001-03-05 |
WO2001011051A3 (de) | 2001-06-21 |
JP2003506089A (ja) | 2003-02-18 |
CA2379165A1 (en) | 2001-02-15 |
EP1198571A2 (de) | 2002-04-24 |
DE10082298D2 (de) | 2001-11-08 |
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